Genetically stable attenuated polioviruses comprising multiple mutations in domain V of the 5′ noncoding region

ABSTRACT

The invention provides an attenuated poliovirus which does not have a base pair mismatch in stem (a) or (b) of domain V of the 5′ non-coding region of its genome, wherein at least seven of the base pairs in stems (a) and (b) are U-A or A-U base pairs.

This application is a national phase filing under 35 USC §371 of PCTInternational Application Serial No. PCT/GB2007/003065, filed Aug. 10,2007, which claims priority to GB Patent Application Serial No.0615933.9, filed Aug. 10, 2006, both of which applications areincorporated by reference herein in their entirety.

FIELD OF THE INVENTION

This invention relates to attenuated polioviruses, to their preparationand to vaccines containing them. More specifically, the inventionrelates to polioviruses which are attenuated and genetically stabilisedby the introduction of defined mutations into their genomes. Thesepolioviruses are particularly useful as inactivated poliovaccine seeds.

BACKGROUND OF THE INVENTION

The live attenuated poliovirus vaccines developed by Sabin in the 1950shave found great use throughout the world. Vaccine strains derived fromeach of the three poliovirus serotypes, known as Sabin types 1, 2 and 3,were prepared by passage of wild-type viruses in cell cultures and wholeanimals until attenuated strains were obtained. These attenuated virusesare substantially less able to cause poliomyelitis in humans than theoriginal wild-type strains. They are administered orally and replicatein the gut to induce a protective immune response.

Although the live oral poliovirus vaccines are generally regarded assafe, their use is associated with a small incidence of paralysis invaccinees. This is most often associated with type 2 and 3 serotypes andrarely, if ever, with type 1. Efforts have, therefore, been made todevelop improved type 2 and type 3 vaccines which would be at leastcomparable in safety to the excellent type 1 strain.

The Sabin vaccine strains were developed by essentially empiricalprocedures. The genetic basis of their attenuation is not completelyunderstood. Over the past several years, however, scientists haveemployed a number of molecular biological techniques in an attempt toelucidate the mechanism by which the neurovirulence of these vaccinestrains is reduced. Most of the work has concentrated on serotypes 1 and3. For both of these the complete nucleotide sequences of the vaccinestrains have been compared with those of their neurovirulentprogenitors.

In the case of poliovirus type 1, the vaccine strain differs from itsprogenitor at 47 positions in the 7441 base genome (Nomoto et al., Proc.Natl. Acad. Sci. USA 79:5793-5797, 1982). All of these are simple pointmutations and 21 of them give rise to amino acid changes in virus-codedproteins. Although several mutations are thought to contribute to theattenuation phenotype of the vaccine strain, direct evidence has beenpresented that the mutation of A-G at position 480 in the 5′ non-codingregion of the genome has a marked attenuating effect on the virus(Nomoto et al., UCLA Symp. Mol. Cell. Biol., New Series, 54 (Eds M. A.Brinton and R. R. Rueckert):437-452, New York: Alan R. Liss Inc.,1987)).

Analogous studies on poliovirus type 3 reveal just 10 nucleotidesequence differences in the 7432 base genome between the vaccine and itsprogenitor strain (Stanway et al., Proc. Natl. Acad. Sci. USA81:1539-1543, 1984). Just three of these give rise to amino acidsubstitutions in virus-encoded proteins. The positions of bases in the5′ non-coding region of the genome of type 3 poliovirus are numberedherein according to the numbering system of Stanway et al., 1984.

The construction of defined recombinants between the type 3 Sabinvaccine strain and its progenitor strain has allowed the identificationof the mutations which contribute to the attenuation phenotype. One ofthese is at position 2034 and causes a serine to phenylalanine change invirus protein VP3.

The other mutation of interest is C (progenitor) to U (vaccine strain)at position 472 in the 5′ non-coding region of the genome. This 472 Umutation has been observed to revert to the progenitor (wild-type) 472 Crapidly upon replication of the virus in the human gut (Evans et al.,Nature 314:548-550, 1985). This reversion is associated with an increasein neurovirulence. C at position 472 has also been shown to be essentialfor growth of a mouse/human polio recombinant virus in the mouse brain(La Monica et al., J. Virol. 57:515-525, 1986). More recently, it hasbeen observed that A changes to G at position 481 in poliovirus type 2,again upon replication of the virus in the gut of vaccinees (Macadam etal., Virology 181:451-458, 1991).

A model for the secondary structure of the 5′ non-coding region of thegenome of poliovirus type 3 Leon strain has previously been proposed(Skinner et al., J. Mol. Biol. 207: 379-392, 1989). As concerns domain V(nucleotides 471-538), bases at positions 471-473 and 477-483 are pairedwith bases at positions 538-536 and 534-528 respectively as follows:

      471         477       483 . . . U C C . . . C C A U G G A . . .. . . A G G . . . G G U G C C U . . .       538         534       528

For convenience, the paired regions are termed stem (a)(471-473/538-536) and stem (b) (477-483/534-528). Previously, we foundthat a type 3 poliovirus with the base pair 472-537 reversed, i.e. 472 Gand 537 C, is attenuated. Further, this attenuated virus had a slightlylower LD₅₀ value than the corresponding poliovirus which only had themutation C to G at position 472 but which retained the wild-type G atposition 537. Attenuated polioviruses in which a base pair of stem (a)or stem (b) of domain V is reversed are disclosed in EP-A-0383433.However, subsequent experiments showed that the type 3 poliovirus inwhich the 472-537 base pair is reversed is not as attenuated as the type3 Sabin vaccine strain.

We have also reported previously the production of attenuatedpolioviruses which have substantially the same attenuation as, orgreater attenuation than, the Sabin vaccine strain (so that they aresafe to use) but which are much more stable genetically. Theseattenuated polioviruses do not have a U-G base pair or other base pairmismatch in stem (a) or (b) of domain V of the 5′ non-coding region ofthe poliovirus genome. (A departure from Watson-Crick base pairing isconsidered to be a mismatch.) More specifically, we prepared type 3polioviruses which contained the following U-A base pairs:

(a) S15: U-A at 472-537, U-A at 480-531 and U-A at 481-530; or

(b) S16: U-A at 472-537, U-A at 480-531 and A-U at 482-529.

Under conditions which rapidly selected neurovirulent variants of Sabin3, the attenuation phenotypes of these poliovirus strains were stable(WO98/41619).

As a result of the success of the global polio eradication programme theproportion of cases attributable to vaccine-derived strains hasincreased dramatically and will continue to do so until live virusvaccination ceases. Partly in response to this, many developed countrieshave already switched to inactivated poliovaccines (IPV) which arecurrently produced from wild strains. When wild-type polio is eradicatedwild-type strains will require high levels of biological containment,which may not be easy to reconcile with the production scales requiredfor IPV, making the use of attenuated vaccine strains for IPVmanufacture attractive, though it has been argued that both wild andattenuated strains ultimately present the same containment issues.

There remains a need for poliovirus strains that are non-infectious forhumans at exposure levels potentially encountered in vaccine productionfacilities. This would significantly reduce the likelihood of escapeinto the environment and the consequences of escape would be negligibleeven after live virus vaccination has ceased. Such strains may be grownunder containment levels that are not prohibitive for vaccinemanufacturers.

SUMMARY OF THE INVENTION

We have now designed and constructed poliovirus strains that may solvethe safety and containment problems of current IPV seeds. These strainsgrew to titres as high as those of Sabin strains in cell culture at 33°C. but infectivity at 37° C. was significantly reduced, and by more thanone million-fold in one case. This type 3 strain appeared completelyattenuated, causing no clinical symptoms at all when inoculatedintraspinally into TgPVR mice at a dose 5,000 times higher than the doseof Sabin 3 required to paralyse 50% of mice. The strains are alsodesigned to be genetically stable using an approach involvingmanipulation of RNA secondary structure in domain V of the 5′ non-codingregion. Sequences of capsid proteins are unchanged in these strains soimmunogenicity of inactivated preparations is expected to be unimpaired.

Accordingly, the present invention provides an attenuated polioviruswhich does not have a U-G base pair or a base pair mismatch in stem (a)or (b) of domain V of the 5′ non-coding region of the poliovirus genomeand wherein at least seven of the base pairs in stems (a) and (b) areU-A or A-U base pairs. Preferably, at least five of the base pairs instem (b) are U-A or A-U base pairs. The poliovirus may be a type 1, type2 or type 3 poliovirus. An attenuated type 3 poliovirus in which the 5′non-coding region of the genome of the poliovirus contains a U-A basepair at positions 472-537, 478-533, 480-531 and 481-530 is preferred.Particularly, preferred are attenuated polioviruses which additionallycontain an A-U base pair at position 482-529 or 477-534, or both.“Attenuated” means attenuated with respect to the wild-type polioviruswhich is the progenitor of the relevant Sabin vaccine strains (eachstrain has its own progenitor) and also with respect to the relevantSabin vaccine strain. Overall, the virus must be sufficiently attenuatedto be non-infectious for humans.

The present invention also provides:

a poliovirus of the invention which is inactivated;

a poliovirus of the invention for use in a vaccine;

a vaccine comprising a poliovirus of the invention and apharmaceutically acceptable carrier or diluent;

use of a poliovirus of the invention as an inactivated poliovaccineseed; and

a method for preparing an inactivated poliovaccine, comprising:

-   -   (i) growing an attenuated poliovirus according to the invention;    -   (ii) inactivating said poliovirus; and    -   (iii) formulating said inactivated poliovirus with a        pharmaceutically acceptable carrier or diluent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the predicted RNA secondary structure of domain V(nucleotides 471-538) of the type 3 Sabin strain (SEQ ID NO: 1). Thebase-paired stem region from 471-473 and 536-538 is stem (a) and thebase-paired stem region from 477-483 and 528-534 is stem (b).

FIG. 2 shows the sequence of stems (a) and (b) of domain V of the Sabinvaccine strains of each type of poliovirus. Domain V of a type 3poliovirus extends from positions 471-538. Domain V of a type 2 or atype 1 poliovirus extends from positions 468-535.

FIG. 3 shows the predicted RNA secondary structure of domain V(nucleotides 471-538) of the prior art attenuated poliovirus straindesignated S15 (SEQ ID NO: 2).

FIG. 4 shows the predicted RNA secondary structure of domain V(nucleotides 471-538) of the attenuated strain of the inventiondesignated S17 (SEQ ID NO: 3).

FIG. 5 shows the predicted RNA secondary structure of domain V(nucleotides 471-538) of the attenuated strain of the inventiondesignated S18 (SEQ ID NO: 4).

FIG. 6 shows the predicted RNA secondary structure of domain V(nucleotides 471-538) of the attenuated strain of the inventiondesignated S19 (SEQ ID NO: 5).

FIG. 7 shows the results of temperature sensitivity tests usingpoliovirus strains, Sabin 3, S15, S17 and S18. The reduction in numberof plaques compared to the number of plaques at 33° C. is shown as afunction of temperature, when grown in L20B, Vero and Hep2C cells.

FIG. 8 shows the one step growth curves of Sabin 3 (S3), S15, S17 andS18 when grown on HEp2C cells at 33° C. TCID₅₀: tissue cultureinfectious dose 50%.

FIG. 9 shows the stability of attenuating mutations in domain V of Sabin2 and Sabin 1 on passage in different cell lines. Viruses were passagedten times in different cells at 37° C. then mutant proportions weremeasured by PCR and restriction endonuclease digestion (MAPREC). (A)Mutation at nucleotide 481 in Sabin 2 during passage in L20B cells (▴)and Vero cells (▪). (B) Mutation in Sabin 1 during passage in Vero cellsat nucleotides 480 or 525 (▴) and at nucleotide 476 (▪). (C) Mutation inSabin 1 during passage in L20B cells at nucleotides 480 or 525 (▴) andat nucleotide 476 (▪).

FIG. 10 shows the stability of 472U in Sabin 3 on passage in differentcell lines. Sabin 3 was passaged ten times in different cells at 37° C.472C content was then measured by PCR and restriction endonucleasedigestion (MAPREC). Symbols ♦, L20B cells; ●, Vero cells: ▴ MRC-5 cells.L20B cells are mouse L cells expressing the human poliovirus receptor.Vero cells and MRC-5 cells are used for vaccine production.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an attenuated poliovirus which does not have abase pair mismatch in stem (a) or (b) of domain V of the 5′ non-codingregion of its genome, wherein at least seven of the base pairs in stems(a) and (b) are U-A or A-U base pairs. Preferably, at least five, suchas six or seven, of the base pairs in stem (b) are U-A or A-U basepairs. Preferably, at least two of the base pairs in stem (a) are U-A orA-U base pairs, for example, three of the base pairs in stem (a) may beU-A or A-U base pairs.

Attenuated polioviruses of the invention have been modified so thatstems (a) and (b) of the domain V do not contain a U-G base pair orother base pair mismatch such as the U-U mismatch in the type 1 Sabinvaccine strain. Preferably, stems (c) and (d) also do not contain a U-Gbase pair or other base pair mismatch. An alternative A-U or U-A basepair is provided in place of the pair mismatch. Thus, a U-A base pair ispreferably present at positions 472-537 and 480-531 of domain V of atype 3 poliovirus, and at position 527-478 of a type 2 poliovirus,replacing U-G base pairs (refer to FIG. 2).

In addition, stems (a) and/or (b) of domain V have been modified toreplace two or more G-C or C-G base pairs with A-U or U-A base pairs.Stem (b) of Sabin 3 contains C-G base pairs at positions 477-534 and478-533 and G-C base pairs at positions 481-530 and 482-529. Two, threeor four of these base pairs are replaced with U-A, A-U or a mixture ofA-U and U-A base pairs. Stem (a) may also be modified to replace the C-Gbase pair at position 473-536 with an A-U or U-A base pair, preferablyan A-U base pair.

In one embodiment, an attenuated poliovirus according to the inventioncomprises domain V of the 5′ non-coding region of poliovirus type 3 inwhich a U-A base pair is present at position 472-537 in stem (a) and atpositions 478-533, 480-531 and 481-530 in stem (b). A further A-U basepair may be present at position 482-529 in stem (b) and/or at position477-534 in stem (b).

Type 1 and type 2 polioviruses can be correspondingly derived from thesequence of stems (a) and (b) of the wild-type neurovirulent type 1 andtype 2 polioviruses. All strains are preferably Sabin. Alternatively,the entire domain V from a type 3 poliovirus of the invention mayreplace the entire domain V from a type 1 or type 2 poliovirus. Forexample, the entire 5′ non-coding region from a type 3 poliovirus of theinvention may replace the entire 5′ non-coding region from a type 1 ortype 2 poliovirus.

The mutations in the polioviruses of the invention attenuate thevirulence of the virus and genetically stabilise existing liveattenuated vaccine virus strains, thereby making them less likely torevert to virulence. These mutations also make the virus safe to produceat a lower containment level than the containment level required for thewild-type viruses used to produce inactivated poliovaccines and thecontainment level that would be necessary to grow the existingattenuated Sabin strains for inactivated poliovaccine production.

An attenuated poliovirus according to any one of the preceding claimsmay be inactivated.

The present invention provides a process for the preparation of anattenuated poliovirus of the invention, which process comprises:

-   -   (i) introducing the or each desired mutation by site-directed        mutagenesis into a sub-cloned region, which includes the or each        position it is wished to mutate, of a DNA copy of a poliovirus        genome;    -   (ii) reintroducing the thus modified region into a complete copy        DNA from which the region was derived; and    -   (iii) obtaining live virus from the copy DNA thus obtained.

A mutation can thus be introduced into a strain of a poliovirus,normally a Sabin strain, by site-directed mutagenesis of a copy DNAcorresponding to the genomic RNA of a poliovirus. This may be achievedby sub-cloning an appropriate region from an infectious DNA copy of apoliovirus genome into the single strand DNA of a bacteriophage such asM13.

After the introduction of the or each mutation, the modified sub-clonedcopy DNAs are reintroduced into the complete copy DNA from which theywere derived. Live virus is recovered from the mutated full length copyDNA by production of a positive sense RNA typically using a T7 promoterto direct transcription in vitro (Van der Werf et al., Proc. Natl. Acad.Sci. USA 83:2330-2334, 1986).

The recovered RNA may be applied to tissue cultures using standardtechniques (Koch, Curr. Top. Microbiol. Immunol. 61:89-138, 1973). Aftertwo to three days of incubation, virus can be recovered from thesupernatant of the tissue culture. The level of neurovirulence and thusof attenuation of the modified virus may then be compared with that ofthe unmodified virus using a standard LD₅₀ test in mice or theabove-mentioned WHO-approved vaccine safety test in monkeys.

Attenuation due to weakening of domain V has also been shown tocorrelate approximately with temperature sensitivity in BGM cells(Macadam et al., Virology 181:451-458, 1991) or in L20B cells (asdescribed for CM-1 cells in Macadam et al., Virology 189:415-422, 1992).The temperature sensitivity of modified virus can thus be determined asa preliminary screen to determine the level of attenuation expected.This can be expressed as the temperature (T) at which the number ofplaque forming using (pfu) is reduced by a power of 10 (1.0 log₁₀) fromthe number obtained at, for example, 33° C. or 35° C. in the same cells.The lower the value of T, the greater the degree of attenuation.

The attenuated polioviruses can be used as live vaccines. They may,therefore, be formulated as pharmaceutical compositions furthercomprising a pharmaceutically acceptable carrier or diluent. Any carrieror diluent conventionally used in live vaccine preparations may beemployed. For example, the attenuated polioviruses can be stabilised in1M aqueous MgCl₂ and administered as a mixture of the three serotypes.

The attenuated polioviruses can, therefore, be used to preventpoliomyelitis in a human patient. For this purpose, they may beadministered orally, as a nasal spray, or parenterally, for example bysubcutaneous or intramuscular injection. A dose corresponding to theamount administered for a conventional Sabin vaccine strain, such asfrom 10⁴-10⁶ TCID₅₀, may be administered.

The attenuated polioviruses may be used as inactivated-poliovaccine(IPV) seeds. Accordingly, the present invention provides an inactivatedattenuated poliovirus of the invention and the use of a poliovirusaccording to the invention as an inactivated poliovaccine (IPV) seed.Also provided by the invention is a method for preparing an inactivatedpoliovaccine, comprising:

-   -   (i) growing an attenuated poliovirus according to the invention;    -   (ii) inactivating said poliovirus; and    -   (iii) formulating said inactivated poliovirus with a        pharmaceutically acceptable carrier or diluent.

The poliovirus may be inactivated by any suitable method. Typically,methods used to inactivate wild-type poliovirus in the currently usedIPVs are employed. For example, the poliovirus may be inactivated byformaldehyde treatment.

Attenuated poliovirus strains of the invention may be inactivated andcombined with a pharmaceutically acceptable carrier or diluent. Anycarrier or diluent conventionally used in inactivated viruspreparations, such as IPV preparations, may be employed. The IPVpreparation may comprise inactivated type 1, type 2 and type 3polioviruses.

The attenuated inactivated polioviruses of the invention can thereforebe used to vaccinate against poliomyelitis in a human patient. For thispurpose, they may be administered by any suitable route, such asparenterally. Parenteral administration may be by subcutaneous orintramuscular injection. A dose corresponding to the amount administeredfor a conventional IPV, such as 8 to 40 D antigen units, may beadministered.

The following Examples illustrate the invention.

Examples

Construction and Recovery of Site-Directed Mutants

S15, S17, S18 and S19 are derivatives of the type 3 oral poliovaccinestrain Sabin 3. Derivation of the Sabin 3 cDNA clone and construction ofS15 have been described previously (Westrop et al, J. Virol.63:1338-1344, 1989; WO 98/419619). Mutated nucleotides are shown in boldin FIGS. 3 to 6, otherwise sequences are identical to Sabin 3.Replacement of C-G base-pairs by U-A or A-U base-pairs progressivelylowers the thermodynamic stability of domain V; removal of all U-Gbase-pairs makes the structure genetically stable as any single mutationwould then weaken the relevant base-pair. Two simultaneous mutationswould be required to strengthen the structure as this could only beachieved by changing a U-A base-pair to a C-G (or G-C) base-pair.

Viruses were constructed and recovered by standard methods. Morespecifically, S17, S18 and S19 were constructed by PCR mutagenesis. Foreach plasmid, three fragments of the 5′ non-coding region of Sabin 3were amplified by PCR using primers incorporating the necessary sequencechanges (as shown in FIGS. 4 to 6), located at nucleotides (a) 31-50 and471-489, (b) 471-489 and 522-540 and (c) 522-540 and 755-778. The threeoverlapping fragments (a)-(c) were gel-purified, mixed and re-amplifiedwith outer primers then the 747 bp fragment comprising the mutated 5′non-coding region was cloned into pCR2.1 (Invitrogen) and sequenced.M1uI-SacI (279-751) fragments with correct sequences were ligated intoSabin 3 clones lacking the SacI-SacI (751-1900) fragment. Full-lengthinfectious clones were generated by addition of a partial SacI/SmaI(2768) fragment.

Under conditions which rapidly selected neurovirulent variants of Sabin3, the attenuation phenotypes of poliovirus strain S15 and S16 werestable (WO98/41619). In order to generate genetically stable strains ofall three serotypes the entire 5′ non-coding region of Sabin 1 wasreplaced exactly with that of strain S15 to create S15/1 and the entire5′ non-coding region of Sabin 2 was replaced exactly with that of strainS15 to create S15/2.

More specifically, to make the S15/1, the 5′ non-coding region of S15was spliced precisely onto the coding region of Sabin 1 by PCRmutagenesis. The start of the coding region of the Sabin 1 clone pT7/S1Fwas amplified by PCR, digested with SacI and AatII, and gel purified.Plasmid pT7/S15 was digested with EcoRI and SacI, and the 0.78-kbfragment containing the T7 promoter and the first 751 nucleotides of thegenome was gel purified. These fragments were ligated together intoEcoRI-AatII-digested pT7/S1F to produce the full-length plasmid clonepT7/S15/1, which contained the entire 5′ NCR of S15 and the codingregion and 3′ NCR of Sabin 1, as verified by sequencing of the first1,200 nucleotides of the genome. As a consequence of the mutagenesisstrategy, a silent T→A change was introduced into the second codon ofthe coding region of pT7/S15/1 compared to Sabin 1.

To make S15/2, the 5′ non-coding region of T7/S15 was spliced preciselyonto the coding region of Sabin 2 by overlapping PCR. The 5′ NCR ofpT7/S15 and the start of the coding region of the Sabin 2 clone pS2 wereamplified; the overlapping fragments were gel purified, mixed, andreamplified with outer primers NP7 and AM13; and the resulting fragmentswere digested with NotI and SacI, gel purified, and ligated intoNotI-SacI-digested pS2 to produce the full-length plasmid clonepT7/S15/2, which contained the entire 5′ NCR of S15 and the codingregion and 3′ NCR of Sabin 2, as verified by sequencing of the first1,500 nucleotides of the genome. Other than the exchanged 5′ non-codingregion, no mutations were introduced into the Sabin 2 sequence.

Two further S18 strains comprising sequences from the poliovaccinestrains Sabin 1 (S18/1) and Sabin 2 (S18/2) were also constructed. S18/1was generated by swapping the 0.78 kb EcoRI-SacI fragment of S18,containing the T7 promoter and the first 751 nucleotides of the genome,into S15/1. S18/1 comprises the 5′ non-coding region of S18 splicedprecisely onto the coding and 3′ non-coding regions of Sabin 1. To makeS18/2 the MluI-BamHI (674) fragment of S18 was swapped into a sub-cloneof S15/2 then the full-length clone was generated using unique MluI andSacI (1318) sites in S15/2. S18/2 comprises the 5′ non-coding region ofS18 spliced precisely onto the coding and 3′ non-coding regions of Sabin2.

Viruses were recovered by transfection of HEp2C monolayers with ≧2 μg T7transcripts (Van der Werf et al, Proc. Natl. Acad. Sci. USA83:2330-2334, 1986) followed by incubation at 33° C. for 24-48 hours, bywhich time complete cytopathic effect was apparent. Sequences of 5′non-coding regions of all mutants were confirmed following RNAextraction and RT-PCR.

Temperature Sensitivity

We have previously shown that for genetically defined poliovirus strainsthat differ only in RNA domain V of the 5′ non-coding region,temperature-sensitivity of growth is quantitatively related to thepredicted stability of the folded RNA (Macadam et al., Virology189:415-22, 1992).

Temperature-sensitivity assays were carried out using L20B, Hep2C andVero cells as described in the above publication. Briefly, viruses wereassayed by plaque-formation at different temperatures. These werecontrolled by incubation of inoculated cell culture plates in sealedplastic boxes submerged in water baths whose temperatures fluctuated by<0.01° C. Graphs in FIG. 7 show curves representing reduction in numbersof plaques compared to 33° C. as a function of temperature in threedifferent cell lines

Results show that weakening of domain V RNA secondary structure has asignificant impact on the ability of the virus to replicate at humanbody temperature in all cell lines tested. In L20B cells there was noevidence of replication at all of S18 at 37° C. even using inoculacontaining 5×10⁵ infectious units.

One-Step Growth Curves

Replicate HEp2C cell sheets were infected synchronously with thedifferent viruses at a multiplicity of infection of 10, incubated at 33°C. for different periods then harvested by freezing at −70° C. Virustitres in cell lysates were determined by standard methods (at 33° C.).Replication kinetics and virus yields were not significantly differentfor any of the viruses. The results are shown in FIG. 8.

Attenuation Phenotypes

Over the last 15 years the use of transgenic mice expressing the humanpoliovirus receptor to assess virulence of polioviruses has beenestablished and validated.

Intraspinal inoculation of transgenic mice expressing the poliovirusreceptor (TgPVR mice) is a highly sensitive method of measuringinfectivity in vivo since virus replication leads to neuronal loss andobvious clinical signs of paralysis. Fewer than ten PFU of wild typeviruses is usually sufficient to paralyse 50% of the mice using thisroute of inoculation (Chumakov et al, Dev. Biol. (Basel) 105:171-177,2001). Here we made use the Tg66-CBA strain of mice, which isparticularly sensitive to type 3 strains, to assess the infectivity ofviruses S15, S17, S18 and S19.

Sabin 3, S15, S17 and S18 viruses were assayed by two routes ofdiffering sensitivity, the intramuscular route and the intraspinalroute. Initial results using these viruses are shown in Table 1. Bothsets of initial experiments showed that S17 and S18 were more attenuated(less virulent) than the current type 3 vaccine strain and S15. S18appeared completely attenuated, causing no clinical symptoms at all wheninoculated intraspinally into the mice at a dose more than 3,000× higherthan the PD₅₀ of Sabin 3.

Further tests using Sabin 1, Sabin 2, Sabin 3, S15, S17, S18, S19, S18/1and S18/2 were carried out using the intraspinal route. The results ofthese tests are shown in Table 2. Strain S15 was indistinguishable fromSabin 3 in these tests (Table 1). Results for strain S17 showed that oneextra C-G to U-A base-pair exchange increased the PD₅₀ more than3000-fold. Strains S18 and S19 have one and two more C-G to U-A (or A-U)exchanges compared to S17 and appeared completely attenuated even atdoses nearly 100.000-fold higher than the PD₅₀ of Sabin 3.

The PD₅₀ of S18/1 was more than a million-fold higher than that of Sabin1 (Table 2), simply as a result of the 5′ non-coding region exchange.The data for S18/1 are consistent with two extra C-G to U-A base-pairexchanges increasing PD₅₀ values by more than 10⁶-fold.

Sabin 2 is the most attenuated of the three oral poliovaccine strainsand has a relatively high PD₅₀ in TgPVR mice (Dragunsky et al, Bull.World Health Organ. 81:251-60, 2003). The PD₅₀ of S18/2 by theintraspinal route was even higher than that of Sabin 2 in Tg66-CBA mice(Table 2). Data for strains S18 and S18/1 suggests it would be severalorders of magnitude higher than 10^(8.1) but it was impractical togenerate a virus preparation of high enough titre to test this.

Genetically Stable Strains of all Three Poliovirus Serotypes

The stabilities of the S15/1 and S15/2 strains were compared with thoseof the relevant Sabin strains in the same way as for strain S15 usingcell culture models which favoured rapid reversion at attenuatingnucleotides (FIG. 10).

The major attenuating mutation in the 5′ non-coding region of Sabin 2 isan A at nucleotide 481 (equivalent to nucleotide 484 in FIG. 1) and an Ato G mutation at this position, which results in significant loss ofattenuation, was rapidly selected during passage of Sabin 2 in both L20Bcells and Vero cells at 37° C. (FIG. 9A). By the third passage in L20Bcells over 60% of the Sabin 2 population had a G at nucleotide 481 andafter four passages selection was almost complete. In Vero cells over60% of the Sabin 2 population had a G at nucleotide 481 after fivepassages and selection was essentially complete after 8-9 passages. Nonucleotide changes were observed in domain V of strain S15/2 after tenpassages in either L20B cells or Vero cells.

Three different mutations in domain V of Sabin 1 are selected duringreplication in the human gut, all of which strengthen base-pairing: 480G to A, 525 U to C and 476 U to A (see FIG. 2). Nucleotides 480 and 525form a base-pair so mutations occur at one or other position in a virusbut not both. In Vero cells mutations at all three positions wereselected in Sabin 1 at a steady rate (FIG. 9B) so that after sixpassages half of the virus population had a mutation at either 480 or525 and more than 40% of the virus population had a mutation at 476.During the last four passages the proportion of the virus populationthat had a mutation at either 480 or 525 increased to approximately100%, mainly due to mutation at nucleotide 480. Mutations at all threepositions were also selected in Sabin 1 during passage in L20B cells(FIG. 9C), although, in contrast to results in Vero cells, mutations at476 selected at a higher rate than those at 480 and 525 so that aftersix passages approximately 60% of the virus population had a mutation atnucleotide 476 and 30% had a mutation at either 480 or 525. Nonucleotide changes were observed in domain V of strain S15/1 after tenpassages in either L20B cells or Vero cells.

TABLE 1 Attenuation/neurovirulence phenotypes in TgPVR mice Log₁₀ PD₅₀values in TgPVR mice i.m. i.s. Sabin 3  9.65  3.6 S15  8.9 n.d. S17 >9.3(0/8) ¹ >7.1 (1/8) S18 >9.4 (0/8) >7.1 (0/8) i.m. intramuscular routei.s. intraspinal route n.d. not determined PD₅₀ paralytic dose (50%) ¹(proportion of mice paralysed at maximum dose)

TABLE 2 Attenuation/neurovirulence phenotypes in TgPVR mice Virus PD₅₀i.s./log₁₀ CCID₅₀ Sabin 1  2.25 S18/1 >8.6 (1/16)* Sabin 2  6.4S18/2 >8.1 (0/8)* Sabin 3  3.6 S15  3.7 S17 >7.1 (4/16)* S18 >8.4(0/16)* S19 >8.2 (0/16)* i.s. intraspinal route PD₅₀ paralytic dose(50%) *paralysed/total at highest dose

The invention claimed is:
 1. A poliovirus which does not have a basepair mismatch in stem (a) or (b) of domain V of the 5′ non-coding regionof its genome, wherein at least seven of the base pairs in stems (a) and(b) are U-A or A-U base pairs.
 2. A poliovirus according to claim 1,wherein at least five of the base pairs in stem (b) are U-A or A-U basepairs.
 3. A poliovirus according to claim 1, wherein six of the basepairs in stem (b) are U-A or A-U base pairs.
 4. A poliovirus accordingto claim 1, wherein seven of the base pairs in stem (b) are U-A or A-Ubase pairs.
 5. A poliovirus according to claim 1, wherein two of thebase pairs in stem (a) are U-A or A-U base pairs.
 6. A poliovirusaccording to claim 1, wherein three of the base pairs in stem (a) areU-A or A-U base pairs.
 7. A poliovirus according to claim 1, whichcomprises domain V of the 5′ non-coding region of poliovirus type 3 inwhich a U-A base pair is present at position 472-537 in stem (a) and atpositions 478-533, 480-531 and 481-530 in stem (b).
 8. A poliovirusaccording to claim 7, in which an A-U base pair is present at position482-529 in stem (b).
 9. A poliovirus according to claim 7, in which anA-U base pair is present at position 477-534 in stem (b).
 10. Apoliovirus according to claim 1 which is inactivated.